Immunologic interactions between intact pathogens and their released soluble antigens
During infections with Streptococcus pneumoniae (Pn), as well as other extracellular bacteria, the immune system likely encounters a variety of microbial components in soluble form, as well as those associated with the intact bacterium (1, 2). Thus, secreted hydrolases such as hyaluronidases, neuraminidases, and endoglycosidases can mediate bacterial spread and destruction of host tissue through degradation of hyaluronan, mucins, and glycolipids. In addition, during the stationary growth phase, Pn expresses a major autolysin (LytA amidase) which degrades it's own peptidoglycan cell wall, resulting in release of cytoplasmic proteins (3, 4). One such protein is pneumolysin that can induce host cell injury through formation of cell membrane pores (5), and at lower concentrations stimulate release of pro-inflammatory mediators (6) and directly accelerate cell death of neutrophils (7), the major phagocytic cell that mediates innate immunity to extracellular bacteria. In addition, since both capsular polysaccharide (PS) and a number of proteins are covalently attached to the bacterial cell wall peptidoglycan (8, 9), the release of soluble PS-protein conjugates upon bacterial lysis is also likely.
Adaptive immunity to extracellular bacteria is largely mediated by antibody. Although soluble and particulate antigens may exhibit distinct immunologic properties (10-12), their potential cross-regulatory effects on the humoral immune response, following concomitant immunization, as might occur during bacterial infections, is unknown. In particular, the context in which the antigen is expressed may impact on the manner in which it is transported and/or processed within the secondary lymphoid organ. This, in turn may significantly impact on the quality and quantity of the subsequent immune response. The size of the immunogen (13-15), its soluble or particulate nature (16, 17), the valency (18-20) and biochemical nature of the antigenic epitope (21), and the presence of associated innate immune cell activators, such as TLRs (22, 23), and mediators of cellular uptake, such as scavenger receptor ligands (24-26), in turn can influence the outcome of these processes.
We investigated the immunologic consequences of co-immunization with intact Pn and soluble conjugates of Pn-derived proteins and polysaccharides (PS), as a model (27). Co-immunization of mice i.p. with Pn and conjugate resulted in marked inhibition of conjugate-induced PS-specific memory, and primary and memory anti-protein Ig responses. Inhibition occurred with unencapsulated Pn, encapsulated Pn expressing different capsular types of PS than that present in the conjugate, and with conjugate containing protein not expressed by Pn, but not with 1 mm latex beads in adjuvant. Inhibition was long-lasting, occurred only during the early phase of the immune response, but was not associated with tolerance. Pn inhibited the trafficking of conjugate from the splenic marginal zone to the B cell follicle and T cell area, strongly suggesting a potential mechanism for inhibition. These data suggest that during infection, bacterial-associated antigens are the preferential immunogen for anti-bacterial Ig responses.
We will determine whether local s.c. co-immunization with Pn and soluble antigens trafficking to the draining lymph node results in the same type of inhibition as that observed via systemic co-immunization to the spleen. The ability of other intact bacteria, besides Pn to mediate inhibition, and whether inhibition occurs for both small (~< 70kDa) antigens trafficking through the conduit system and large antigens requiring cell transport will be determined. Most importantly, the mechanism of this inhibition will be explored, including the potential ability of Pn to block cell binding, uptake, and/or trafficking of soluble antigens to sites within the lymphoid compartment critical for elicitation of T cell-dependent Ig responses. Use of both flow cytometric analysis and confocal microscopy to accomplish these goals will be employed. Finally, the potential of Pn to inhibit CD4+ T cell activation in response to soluble antigens, will be determined using a transgenic CD4+ T cell approach.
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